Isolation and culture of cells
For human studies, approval from the Ethics Committee of Dalian Medical University as well as written informed consent from all participants was obtained. The study was performed in accordance with the principles of the Declaration of Helsinki. Human bone marrow mesenchymal stem cells (hBMSCs) were isolated from jawbone removed from patients undergoing orthognathic surgery at the Affiliated Stomatological Hospital of Dalian Medical University. The hBMSCs were cultured and expanded following the established method , in low glucose Dulbecco’s Modified Eagle’s Medium (L-DMEM, Hyclone, Logan, UT, USA), containing 1% penicillin–streptomycin (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and 10% fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific, Inc.) with 5% CO2 at 37°C. hBMSCs up to passage 5 were used in this study.
The isolation of mouse bone marrow mesenchymal stem cells (mBMSCs) was performed as described previously . The mBMSCs were cultured in MEM alpha modification (α-MEM, Hyclone) supplemented with 10% FBS and 1% penicillin-streptomycin. The mBMSCs at passages 3–5 were used for the in vivo study. Human umbilical vein endothelial cells (HUVECs; American Type Culture Collection, ATCC, Manassas, VA, USA) were cultured in endothelial cell medium (ECM, ScienCell, San Diego, CA, USA) with 5% FBS, 1% penicillin-streptomycin, and 1% endothelial cell growth supplement (ECGS, ScienCell) at 37°C with 5% CO2.
Osteogenic differentiation and assay
The cells were divided into three groups: HUVECs, hBMSCs, and co-culture (hBMSCs and HUVECs co-cultured at 5:1) in 24-well plates (5 × 104 cells/well). When the cells reached 100% confluence, the medium was changed to osteogenic medium that comprised α-MEM (Hyclone), supplemented with 10% FBS (Gibco), 10−7 M dexamethasone (Sigma-Aldrich, St. Louis, MO, USA), 10 mM β-glycerophosphate disodium (Coolaber, Beijing, China), and 50 μg/ml L-ascorbic acid (Coolaber). Alkaline phosphatase (ALP) staining, Alizarin Red (ARS) staining, and semi-quantitative analysis were conducted according to the manufacturer’s instructions. To evaluate the ALP, five fields of view were randomly selected under 10× magnification, and then, the images were quantified in the average of optical density by the Image-Pro Plus 7.0 version software (Media Cybernetics, Inc., Rockville, MD, USA). For ARS, the alizarin red was eluted with 10 % cetylpyridinium chloride (CPC, Sigma-Aldrich), and the absorbance was measured at 570nm using a microplate reader (Molecular Devices, Sunnyvale, CA, USA).
Construction of the mBMSCs/BCP complex and the surgical procedure
Biphasic calcium phosphate (BCP), purchased from the Biomaterial Engineering Research Center of Sichuan University (Chengdu, China), was composed of 60% hydroxyapatite (HA) and 40% β-tricalcium phosphate (β-TCP). A porous cylinder of BCP was custom made with a 3-mm diameter× 2-mm height. The porosity was approximately 50%, and the pore size ranged from 300 to 500 μM. To create the mBMSC/BCP complex, mBMSCs (2 × 106/mL) were seeded into BCP sterilized by irradiation and incubated for 3 h for in vivo implantation, or cultured for 7 days for in vitro tests.
Five-week-old C57BL/6 female mice were provided by the Experimental Animal Center of Dalian Medical University. All the experiments were approved by the Institutional Animal Care and Use Committee of Dalian Medical University. A total of 40 mice were randomly divided into four groups: BCP group, BCP alone without LLLT; BCP+LLLT group, the implanted BCP received LLLT every other day for 3 weeks (from 7 days prior to surgery to 14 days post-operation); BCP+mBMSCs group, mBMSCs/BCP without LLLT; and BCP+mBMSCs+LLLT group, the implanted mBMSCs/BCP received LLLT every other day for 3 weeks (from 7 days prior to surgery to 14 days post-operation). For graft implantation, a transverse incision was made at the midpoint of outer canthus line in the mouse. The mucoperiosteal was stripped to the herringbone suture, and the BCP or mBMSCs/BCP was implanted under the calvarial periosteum of the mice at the midpoint of the herringbone suture (posterior fontanel) under anesthesia, and the incision was sutured.
A low energy laser apparatus (GaAlAs semiconductor laser JLT-MD500B, Jinlaite Optoelectronics, Co., Ltd., Wuhan, China) with a continuous wavelength of 808 nm was used in this study. The total energy density of the irradiation spot was measured using a laser power meter. For in vitro LLLT, the cells were irradiated with 4.5 J/cm2 (distance 14 cm, power 100 mW, time 3 min) every day. For in vivo LLLT, the implantation area of the anesthetized mice received LLLT (irradiation dose 1.8 J/cm2, 808 nm; distance 14 cm; power 40 mW; time 3 min; irradiation area 4 cm2). At 1 or 2 months after operation, the implanted grafts were isolated for histological staining.
Hematoxylin and eosin (H&E) and Masson’s trichrome staining (Solarbio, Co., Ltd., Beijing, China) were performed according to the manufacturer’s instructions.
For immunofluorescence staining, the sections were incubated using the primary antibodies (rabbit polyclonal anti-CD31 antibodies, 1:10, ab28364, Abcam; rat monoclonal anti-EMCN antibodies, 1:10, ab106100; Abcam, Cambridge, MA, USA) for 24 h at 4°C. Then the secondary goat anti-rabbit antibodies conjugated with Cy3 568 (1:200 diluted in PBS, ab97075; Abcam) and goat anti-rat conjugated with dylight 488 (1:200 diluted in PBS, A23240; Abbkine, Wuhan, China) were used to incubate the tissues for 1 h at 37°C. Finally, the nuclei were counterstained with 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI, 1:1,000 diluted in PBS, 10236276001; Roche, Basel, Switzerland). The images were photographed under a fluorescence microscope (Olympus Corporation, Tokyo, Japan) and evaluated using Image-Pro Plus version 7.0 software (Media Cybernetics, Inc., Rockville, MD, USA).
For immunohistochemistry, the sections were incubated with primary antibodies (rabbit polyclonal anti-HIF-1α antibodies, 1:400, ab2185, Abcam; rabbit polyclonal anti-TGF-β, 1:200, ab31013, Abcam; mouse monoclonal anti-VEGF, 1:500, ab1316; Abcam) overnight at 4°C, then with the horseradish peroxidase (HRP)-conjugated goat anti-rabbit streptavidin (ZSGB-BIO, Beijing, China) secondary antibody at room temperature for 2 h. Finally, the sections were visualized using 3,3-diaminobenzidine (DAB; ZSGB-BIO), and counterstained with hematoxylin (Solarbio, Beijing, China).
Cell proliferation assay
The effect of LLLT on cell proliferation was assessed by Cell Counting Kit-8 (CCK8, ApexBio Technology, Inc., Houston, TX, USA). According to the manufacturer’s instructions, cells were seeded in a 96-well plate at a density of 2×103 cells / well in 100 μL of culture medium with LLLT (0 J/cm2, 1.5 J/cm2, 4.5 J/cm2, 7.5 J/cm2) for 1-5 days. CCK-8 solution of 10 μL was added to each well of the plate, and then the plate was incubated for 1 hour in the incubator at 37°. Finally, the absorbance was measured at 490 nm using a microplate reader.
Tube formation assay
The cells were seeded into 96-well plates precoated with BD Matrigel™ Matrix (BD Biosciences, Franklin Lakes, NJ, USA) and incubated for 3 h at 37°C. The formed capillary-like structures were fixed and observed under a light microscope (Olympus Corporation). Tube formation was quantified using ImageJ software.
Quantitative real-time polymerase chain reaction (RT-qPCR)
RT-qPCR was performed to evaluate the mRNA expression levels of osteogenesis and angiogenesis genes in HUVECs, hBMSCs, and co-cultured cells. Total RNA was isolated with RNAiso Plus (TRIzol; Takara Bio, Inc., Otsu, Japan) and reverse transcribed to cDNA with HiScript II Q RT SuperMix (Vazyme Biotech, Co., Ltd., Nanjing, China). RT-qPCR was performed using ChamQ Universal SYBR qPCR Master Mix (Vazyme Biotech). GAPDH was used as the internal control for each experiment. The primer sequences for RT-qPCR are listed in Additional file 1: Table S1. Data were analyzed using the 2−ΔΔCt relative expression method.
Protein was isolated from cells in radio immunoprecipitation assay lysis buffer (RIPA, Solarbio) and quantified using a bicinchoninic acid protein assay kit (BCA; Beyotime, Shanghai, China). Then the protein was loaded onto a polyacrylamide gel and electroblotted onto polyvinylidene difluoride (PVDF) membranes (Millipore, Merck KGaA, Darmstadt, Germany). The membrane was blocked with 5% defatted milk and incubated with primary antibody (HIF-1α, 1:1,000; Abcam) at 4°C overnight and then secondary antibody (1:2000; ZSGB-BIO) at room temperature for 1 h. Finally, the protein bands were visualized with a super chemiluminescence detection reagent kit (Thermo Fisher Scientific).
Reactive oxygen species assay (ROS)
ROS were detected using a reactive oxygen species assay kit (Beyotime Biotechnology, Shanghai, China). According to the instructions, cells grown on slices were incubated with serum-free medium containing 2′,7′-dichlorofluorescein-diacetate (DCFH-DA, 10 μM) for 20 min at 37°C. Then the cells were washed with PBS three times and examined under a fluorescence microscope.
All experiments in this study were repeated at least three times. One-way analysis of variance and Student-Newman-Keuls tests were performed using GraphPad Prism 6 (GraphPad Software, La Jolla, CA, USA). The data are presented as means ± SD, and P < 0.05 was considered significant.
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